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Somatic Cell Nuclear Transfer Process

Attempts at cloning a mammal can be traced back to 1979, where the scientist Steen Willadsen successfully cloned a sheep embryo using nuclear transfer [1]. Since then numerous attempts have been made to replicate these results. Notably the birth of Dolly the sheep (1996) was a major development in this field; as she was the first mammal to be cloned from a fully differentiated somatic cell, using somatic cell nuclear transfer (SCNT) [2]. This essay will describe the process of somatic cell nuclear transfer in light of mammalian cloning and the risks it poses to mammalian reproduction.
The fertilization of mammalian gametes through natural reproduction is limited by the ability to preserve desirable traits after the extinction of an individual. Moreover, the reproductive success of natural fertilization is limited by the gestation length, estrus cycle, the efficiency of insemination during intercourse and Hayflick limit [3]. Furthermore, these limitations are chiefly important in livestock agriculture; where desired traits and alleles are more favourable for propagation.
SCNT enables us to extract the nucleus of a fully differentiated somatic cell (diploid cells) and introduce it into an enucleated mature oocyte which is allowed to develop into an embryo; that is genetically identical to the host cell [4]. Other variations to this method are practised even though they all rely on the same principles. By this process, the limitations stated above become insignificant as specific mammals with the desired traits can be cloned to preserve the genome. However, this technique is still undeveloped and the success in producing cloned offspring is low.
The success rate of SCNT is dependent on several factors; namely, selecting the right donor cell that will be most efficient to the nuclear transfer. In this process, fully differentiated somatic cells are selected based on their cell-cycle state and age. The G0 phase is most desired when selecting the donor cell as it has been shown to be the most effectual donor [5]. Conversely, deprivation of nutrient to the donor cells growing in vitro can also induce the cells to adopt the G0 resting phase. The age of donor cells also contribute to the success of cloning, the more aged the donor cell the less efficient SCNT becomes.
Additionally, donor cells that are derived from more genetically diverse species are favoured, as it has been shown that cells obtained from inbred animals are less likely to be successful in cloning [6]. However, these factors are only relative to the limited species that have been examined and more factors may come to light as other species such as primates are subjected to SCNT. Once the donor somatic cells are identified, they are normally extracted from the skin of the donor mammal, using needle aspiration and avoiding unnecessary strain on the donor animal.
Oocytogenesis is the process in which females produce oocytes. SCNT uses mature oocytes in metaphase-ll which are collected from the ovaries of the required animal [7]. The mature oocytes are enucleated using micromanipulation which penetrates the zona pellucida and removes the nucleus.
There are two alternative routes which can be adopted when manipulating the process of the insemination of the nucleus donor cells into the mature oocytes. First, the Honolulu technique (developed by Wakayama) which uses brain cells, cumulus cells and sertoli cells as donors that are naturally in the G0/G1 phase. The nucleus of the somatic cell is aspirated and directly micro-injected into the oocyte using a piezo-impact pipette; which penetrates the zona pellucid and delivers the nucleus into the enucleated oocyte [8]. The oocytes are subsequently activated by exposing them to a medium containing Sr 2 that also contains cytochalasin-B which acts to prevent the formation polar bodies. Figure. 1[9] shows a diagrammatic representation of the Honolulu technique, highlighting that the nucleus is directly inserted into the mature oocyte.
Secondly, the Roslin technique (used to create Dolly the sheep) cultures donor cells in vitro and deprives them of nutrients; forcing the cells to adopt the G0 phase. Subsequently, the enucleated oocyte is aligned next to the donor cell; such that the oocyte and donor cell are parallel to one another. Pulsating electrical currents are applied to fuse the oocyte and donor cell together, by inducing pore formation of the cell membrane [10].
Figure.1In the Honolulu and Roslin techniques the use of chemicals and electrical pulses induce the activation of the oocyte, which can subsequently develop into an embryo which is implanted into a surrogate host for progeny development.
The activation of the oocyte induces major reprogramming of the differentiated donor nuclei back to its totipotent state [11]. This process is extremely intricate and the full biochemical mechanisms are not fully understood. However, extensive research has been completed in understanding an overview of oocyte reprogramming and epigenetic modification. The introduction of a somatic nucleus into the oocyte causes rapid deacetylation of histones on lysine residues, catalysed by histone deacetlase. Moreover, the donor chromatins also experience demethylation [12], which is also a method that is used to dedifferentiate the nuclei back to totipotent state. Aberrant or incomplete DNA reprogramming is thought to be a major contributor to abnormal development in embryos and clones which can explain why only 1% of SCNT are successful in producing fully developed clones.
Figure.2The efficiency of the Honolulu technique and the success rate of cloning have been shown to be superior to the Roslin technique [12]. However, the overall success rate of cloning, irrespective of the method used is still considerably low, with only 1% success rate. Figure. 2 [13] shows the percentage of embryos surviving prior to implantation with surrogate and post implantation.
Moreover, there are several risks associated with clones derived from mammalian SCNT. These risks also have ethical implications that follow.
Phenotypic abnormalities that are associated with clones derived from SCNT ranges from aberrant telomere length (which can lead to premature ageing) to large offspring syndrome and irregular placenta development during embryonic growth.
The telomere length and ageing of clones are thought to be directly correlated. Telomeres are situated on the ends of chromosomes and consist of numerous repetitive DNA bases that function to stabilise and prevent deterioration of the chromosome [14]. Experimental observations show that some species of mammals are prone to shorter telomere lengths in comparison with a control. It is also thought that the telomeres are not fully restored to the original length during SCNT. Such implications can suggest that the sizes of the somatic cell telomeres are inherited by the clones; therefore producing clones that have already aged [15]. Dolly lived until she was 6 years of age (half the age of an average sheep) and was shown to have shorter telomeres in comparison to a control (19 kb vs. 23 kb) implying that she died prematurely. However, shorter telomeres in clones are not universally applicable as in mice, bovine and cattle all showed similar lengths to their respective control, if not longer [16]. The occurrence of shorter telomere lengths in some species suggests that the donor cell species and genetic background govern it. Nevertheless, the exact cause of short telomere length is still not yet fully comprehendible, yet some studies indicate that it might be caused by incomplete reprogramming [17].
Large offspring syndrome (LOS) is characterised by larger than normal clones that have oversized organs and aberrant limb formation which all can lead to an increase in prevalence of organ defects and cardiovascular difficulties. These characteristics have been observed in cattle and can contribute to higher abortions rate and deformities in skeletal structure. However, offspring’s derived from cloned mammals diagnosed with LOS, were shown not to have LOS [18].
This suggests that again irregular epigenetic reprogramming during SCNT is a contributor to LOS as the progeny of the clones (which are born naturally) fail to have LOS.
Embryos that are derived from SCNT have been shown to have abnormal/enlarged placenta development (placentomegaly) during embryonic growth. The abnormalities occur in both bovine and mice [19] and can cause the developing fetus to die during pregnancy. The aberrant placenta in mice is shown to have an increased amount of insulin- like growth factor which can cause LOS in clones. Moreover, failure for the placenta to develop accordingly during the pregnancy of clones can cause immune-mediated abortion [20].
The risks to mammalian reproduction stated above can produce clones that are phenotypically defective which raises ethical concerns. The abnormalities in clones can cause harmful side effects and can lead to cloned mammals suffering. We have seen that some mammals show premature ageing which can ultimately lead to premature death. The welfare of these clones seems to be disregarded in the experiments that are conducted. Moreover, there are concerns that a small proportion of cloned animals can enter our food chain, which is thought to be unsafe. However, recent studies show that consumption of cloned animals is safe to homosapeins [21].
The prospect of human SCNT also has deep ethical implications. Current legislation in all countries prevents SCNT in humans. Nonetheless, the proposed benefits that SCNT offers (therapeutic cloning) may one day outweigh the ethical concerns. If this occurs, it would shake the foundations of tradition, as humans can be ’produced’ asexually with their genomic sequence known [22]. This can lead to ‘gene discrimination by other non cloned humans, and by cooperate companies who can prevent human clones (that may be prone to specific dieses) from obtaining insurance, for example.
In conclusion, Somatic cell nuclear transfer has been successfully used to clone mammals from fully differentiated somatic cell. However, this technique is largely inefficient and a major Impediment is that only 1% of somatic cells successfully developed into clone. The lack of understanding on oocyte reprogramming can be contributed to the inefficiency of this technique. Moreover, this has lead to some clones showing abnormal phenotypic features which has major ethical implications. Nevertheless, somatic cell nuclear transfer shows great promise in the fields of medical therapeutics, agriculture and conservation once all aspects of its process are understood.

Membrane: Structure And Function

Chapter title: Membrane Structure and Function. The “ability of the cell to discriminate in its chemical exchanges with the environment is fundamental to life, and it is the plasma membrane that makes this selectivity possible.”
Membrane
The membranes that are found within cells (plus the plasma membrane surrounding cells) consist of phospholipids (and other lipids plus membrane proteins) arrayed by hydrophobic exclusion into two-dimensional fluids known as known as lipid bilayers
Phospholipids
Phospholipids are amphipathic molecules meaning that they have both a hydrophobic and a hydrophilic end
Lipid bilayer
Phospholipids can exist as bilayers in aqueous solutions
The hydrophobic portion of the phospholipid is shielded in middle of these bilayers
The hydrophilic portion is exposed on both sides to water
Lipid bilayers are held together mainly by hydrophobic interactions (including hydrophobic exclusion)
Fluid mosaic model
The plasma membrane contains proteins, sugars, and other lipids in addition to the phospholipids
The model that describes the arrangement of these substances in and about lipid bilayers is called the fluid mosaic model
Basically, membrane proteins are suspended within a two-dimensional fluid that in turn is made up mostly of phospholipids
Cholesterol
Cholesterol, a kind of steroid, is an amphipathic lipid that is found in lipid bilayers that serves as a temperature-stability buffer
At higher temperatures cholesterol serves to impede phospholipid fluidity
At lower temperatures cholesterol interferes with solidification of membranes (e.g., cholesterol functions similarly, in the latter case, to the effect of unsaturated fatty acids on lipid-bilayer fluidity)
Cholesterol is found particularly in animal cell membranes
Membrane proteins
Proteins are typically associated with cell membranes
Integral membrane proteins are typically hydrophobic where they interact with the hydrophobic portion of the membrane or hydrophilic where they interact with the hydrophilic portion of the membrane and overlying
Functions of membrane proteins
Functions of membrane proteins include:
Transport of substances across membranes
Enzymatic activity
cell communication
Cell-to-cell joining
Attachment to the cytoskeleton and extracellular matrix
Selective permeability
Lipid bilayers display selective permeability
In general, intact lipid bilayers are permeable to:
Hydrophobic molecules (including many gasses)
Small, not-ionized molecules
Simultaneously, lipid bilyaers are NOT permeable to:
Larger, polar molecules (e.g., sugars)
Ions, regardless of size
Thus, lipid bilayers are selectively permeable barriers that allow the entry of small or hydrophobic molecules while blocking the entry of larger polar or even small charged substances
Transport across membranes
Movement across membranes is important, for instance as a means of removing wastes from a cell or bringing food into a cell
Categories of substance transport across membranes include:
Passive transport
Facilitated diffusion
Active transport (including cotransport)
Endocytosis, phagocytosis, and exocytosis, also considered below, technically are not mechanisms of movement of substances across lipid bilayers (though these do represent movements of substances into and out of cells; to be movement across the euakaryotic cell membrane, a substance must actually pass through an endomembrane lipid bilayer)
Note that in considering transport across membranes we will once again confront the concept of movement away from or towards equilibrium, i.e., endergonic and exergonic processes
There are three basic types of movement across membranes: simple diffusion, passive transport, and active transport:
Simple diffusion
Simple diffusion is the movement of substances across lipid bilayers without the aid of membrane proteins
This image (below) shows how substances move through membranes, regardless of net direction and concentration gradients:
This image (below) shows how substances net move through membranes in the direction of their concentrations gradients (i.e., with their concentration gradients)-note that regardless of how net movement is accomplished, all simple diffusion across membranes occurs in the manner illustrated above, i.e., it is a process that is driven by the random movement of molecules:
This figure (below) indicates the kinds of molecules that are capable of moving across membranes via simple diffusion:
Passive transport
Passive transport is the term used to describe the diffusion (as well as what is termed facilitated diffusion, below) of substances across lipid bilayers
Passive transport is a consequence of movement through the lipid bilayer (whether by diffusion through the membrane or with movement across facilitated by an integral membrane protein) a concentration gradient thereby contrasting with active transport
Down the concentration gradient
Diffusion is a random process that tends to result in the net movement of substances from areas of high concentration to areas of low concentration
This includes movement from one side of a permeable lipid bilayer to the other from the higher concentration side to the lower concentration side (i.e., passive transport)
Movement from high to low concentration areas is described as going “down its concentration gradient.”
The direction of movement of substances across lipid bilayers by passive transport is controlled by concentration gradients
Osmosis
Movement of water across selectively permeable membranes down the water concentration gradient is called osmosis
Note that this is movement toward equilibrium (exergonic process)
Tonicity (isotonic, hypertonic, hypotonic)
Picture a membrane separating two solutions, one side with a higher solute concentration than the other
The side with the higher solute concentration is said to be hypertonic
The side with the lower solute concentration is said to be hypotonic
(I keep track of the difference by recalling that a hypodermic syringe is so named because the tip of the needle is placed “beneath” the dermis, i.e., under the skin; a hypotonic solution has a solute concentration that is beneath, i.e., lower than that of the reference solution)
If both sides have the same solute concentration, they are said to be isotonic
Animal cells and tonicity
Normally animal cells are bathed in an isotonic solution
Placement of an animal cell in a hypertonic solution causes the cell to shrink (i.e., water is lost from the cell by osmosis)
Placement of an animal cell in a hypotonic solution causes it to take on water then burst (lyse, i.e., die) (water is gained by the cell, lost from the environment bathing the cell, both by osmosis)
Turgidity
Normally a plant cell exists in a hypotonic environment
The hypotonicity causes the plant cytoplasm to expand
However the plant cell does not lyse and this is due to the presence of its cell wall
This conditions is known as turgidity (i.e., the pressing of the plant plasma membrane up against its cell wall)
Plant cells prefer to display turgidity
Plasmolysis
A plant or bacterial cell placed in a hypertonic environment will show a shrinkage of its cytoplasm
This shrinkage is called plasmolysis
At the very least plasmolysis will inhibit growth
Often plasmolysis will lead to cell death
This is the principle upon which foods are preserved in highly osmotic solutions (e.g., salt or sugar); such solutions impede most microbial growth
Flaccidity
Plant cells bathed in isotonic solutions will fail to display turgidity
Instead they display flaccidity
At a whole-organismal level, flaccidity is otherwise known as wilting
Transport proteins
Substances (e.g., sugars) that are not permeable through lipid bilayers may still cross via membrane-spanning transport proteins
Facilitated diffusion
Facilitated diffusion is the movement of a substance across a membrane via the employment of a transport protein, where net movement can only occur with the concentration gradient, is called facilitated diffusion
The key thing to keep in mind is that facilitated diffusion, in contrast to other mechanisms of transport-protein-mediated membrane crossing, does not require any input of energy beyond that necessary to place the protein in the membrane in the first place (i.e., facilitated diffusion is an exergonic process)
Passive versus active transport
Two general categories of transport across membranes exist:
Those that don’t require an input of energy (passive transport, simple diffusion, facilitated diffusion)
Those that do require an input of energy (active transport)
Passive Transport
Active Transport
Concentration gradient
With (Down)
Against (Up)
Without Integral Protein
Yes (Simple Diffusion)
No
With Integral Protein
Yes
Yes
Examples
Small or Hydrophobic Substances, Osmosis(by simple diffusion) or Not-Small or Charged Substances (by facilitated diffusion)
Cotransport, Proton Pump, Sodium-Potassium Pump
Active transport
Active transport is the movement of substances across membranes against their concentration gradients
Moving things against their concentration gradients requires an expenditure of energy (i.e., it is an endergonic process)
This energy can be in the form of ATP (e.g., sodium-potassium pump)
This energy can also be in the form of electrochemical gradients (i.e., cotransport)
Note that the movement of substances by active transport is in a direction that is away from equilibrium
Sodium-potassium pump
One means by which cells actively transport substances across membranes is via the sodium-potassium pump
The sodium-potassium pump is important especially in animal cells, and is the means by which the sodium-potassium electrochemical gradient is established by these cells
Proton pump
The sodium-potassium pump is the means by which animal cells generate membrane potentials
In bacteria, plants, and fungi, proton pumps play the same role
The proton pump is simply ATP-driven active transport in which the substance pumped across the membrane is a hydrogen ion
Cotransport
Much of the active transport accomplished by a cell isn’t directly powered by ATP
Instead, much active transport is powered by membrane potentials (i.e., electrochemical gradients)
Such electrochemical-gradient-driven active transport is called cotransport
In cotransport, one substance, such as a sugar, is driven up its concentration gradient while a second substance, e.g., sodium ions or protons, are allowed to fall down their electrochemical gradient; the energy gained from the latter is employed to power the former (i.e., energy coupling)
Endocytosis
Endocytosis is a general category of mechanisms that move substances from outside of the cell to inside of the cell, but neither across a membrane (technically) nor into the cytoplasm (again, technically speaking)
Instead, substances are moved from outside of the cell and into the lumens of endomembrane system members
To enter the cytoplasm an endocytosed substance must still be moved across the membrane of the endomembrane system, e.g., following their digestion (typically hydrolysis) to smaller molecules
Examples include: phagocytosis, pinocytosis, and receptor-mediated endocytosis
Phagocytosis
Phagocytosis is the engulfing of extracellular particles is achieved by wrapping pseudopodia around the particles, thus internalizing the particles into vacuoles
Amoebas employ phagocytosis to “eat”
Most protozoa obtain their food by engulfing, i.e., via some form of endocytosis
The advantage of endocytosis as a mechanism of food gathering has to do with minimizing the volume within which digestive enzymes must work in order to digest food, i.e., the engulfed food particle
Cells in our own bodies, called phagocytes and macrophages employ phagocytosis to engulf (and then destroy) debris floating around our bodies as well as to engulf and destroy invading bacteria
Pinocytosis
Pinocytosis is the engulfing of liquid surrounding a cell
This is how developing ova obtain nutrients from their surrounding nurse cells (ova are very large cells so have surface-to-volume problems-pinocytosis solves the problem of nutrient acquisition by allowing nutrients to be obtained across many internal membranes rather than being limited to crossing the plasma membrane)
Receptor-mediated endocytosis
Receptor-mediated endocytosis involves the binding of extracellular substances to membrane-associated receptors, which in turn induces the formation of a vesicles
Exocytosis
Exocytosis is more or less the mechanistic opposite of endocytosis

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